Biochemical And Genetic Basis Of Watermelon Fruit Metabolome

Watermelon is a very important and popular fruit in the world and among the most nutrient-intense fruits.Watermelon fruit can produce and accumulate a large number of metabolites with different physical and chemical properties to maintain plant growth,adapt to the external environment,and fulfill human consumption.However,the metabolomics profiles of natural populations including different types of watermelons have not been studied,and the regulation mechanism of metabolic pathways is rarely reported.In this study,the most comprehensive watermelon fruit metabolomics profile and genetic basis was obtained through genome re-sequencing and wildly targeted metabonomics approach.The watermelon population structure,phylogenetic tree,and stepwise selection of metabolites were analyzed.The important loci were defined through metabolite-based genome-wide association study(mGWAS)analysis,which are related to the metabolites of bitterness,sweet,color,organic acids,and others.The evolutionary relationship between different species in the genus Citrullus was clarified using nucleotide diversity in selective sweeps.The candidate genes related to cucurbitacin were verified.The results revealed the influence of domestication and improvement on watermelon fruit metabolome and provide an important resource for metabolomics-assisted watermelon breeding.This research also provides an effective strategy for study the regulation mechanism of metabolic pathways and crop domestication.1.Large-scale resequencing analysis of watermelon population revealed the genomic variation of watermelon germplasm resources.Genome re-sequencing of watermelon germplasm resources representing different cultivation and domestication status resulted in an average sequencing depth of 9.24 X and an average genome coverage of 86.9%.A total of 4,591,014 SNPs were identified,a comprehensive genome variation map of watermelon was obtained.The population structure and phylogenetic analysis can divide these accessions into different groups.Genetic diversity of wild watermelon was higher than that of cultivars,and the genetic background of cultivated watermelon was narrow.2.The metabonomic differences of different cultivated types of watermelons and the stepwise selection of metabolites during domestication were clarified by wildly targeted metabonomics approach.A total of 3730 metabolic features were identified in the mature fruits of 204 watermelon accessions using widely targeted metabonomics approach,of which 87.1% metabolic features have a CV value greater than 50%,and 50.3% metabolic features with a broad sense heritability value greater than 0.5.The 204 watermelon accessions can be divided into 6 groups through PCA and phylogenetic analysis based on metabolites contents.Edible seed watermelon,a special germplasm type of cultivated watermelon,is grouped into a single category.The levels of fruit cucurbitacins and flavonoids were negatively selected during speciation stage and early stages of domestication,whereas sugar and carotenoids contents were positively selected during domestication stage.Fruit malic-and citric-acid contents were negatively and positively selected respectively,during the later stage of improvement.The modern dessert watermelon was formed by stepwise selection of fruit quality related metabolites in watermelon evolution.3.Metabolite-based genome-wide association study revealed a large number of metabolite-related genetic loci.A total of 2931 significant lead SNPs were detected for 3730 metabolic festures by metabolite-based genome-wide association study analysis,and 23.8% of metabolites were associated with at least one signal.There are 8 loci associsted with cucurbitacins,11 main signals related to carbohydrates,5signals related to citric acid content were detected.Some loci related to sugar and citric acid covered each other during the processes of domestication and improvement.The genome-wide association studies results of flesh color,lycopene content,β-carotenoid content and β-apocarotenal content are co-localized on chromosome 4.The combination of two non-synonymous mutation sites SNP1 and SNP2(SNP1: 4:8886348 and SNP2: 4:8886977)in the lycopene β-cyclase gene can be used to distinguish the red,yellow and white flesh of watermelons accessions.4.The cascade regulatory network and structural gene function verification of cucurbitacin metabolic pathway.A gene cluster in the selective sweep cuc1-3 on chromosome 1 is related to cucurbitacin synthesis.There is a cascade regulation in this gene cluster.ERFs can regulate the transcription levels of bHLHs genes,and bHLHs can regulate the expression of Cla011514,Cla011515,Cla011464 and Cla019330 genes,and ultimately regulate the content of cucurbitacins.Through transient overexpression,virus-induced gene silencing,yeast heterologous expression,prokaryotic protein expression and in vitro enzyme activity test,the function of a 2,3-oxidized squalene synthase gene Cla019330,a glycosyltransferase gene Cla011464 and two P450s(Cla011514 and Cla011515)were verified.Cla019330 gene can catalyze the conversion of 2,3-oxidized squalene to cucurbitadienol.Cla011464 gene can catalyze the glycosylation reaction of cucurbitacin I and cucurbitacin D.The expression of these two P450 genes will affect the accumulation of cucurbitacin I,cucurbitacin E and cucurbitacin B in watermelon.5.The special domestication status of Chinese edible seed watermelon.The metabolomics profile of edible seed watermelon is between that of egusi watermelon and landrace watermelon.The flesh color,sweetness and its related genotypic frequencies in edible seed watermelon was also between that of egusi and landrace watermelon.The results of phylogenetic tree and genetic distance analysis using SNPs in the domestication sweeps related to fruit quality indicates that edible seed watermelon may be a transitional type from egusi watermelon to modern dessert watermelon.These results suggests that C.lanatus edible seed watermelon probably represents the closest relative of the early founder type of watermelon that gave rise to modern dessert watermelons.